1. Field of the Invention
The present invention relates to polypeptides comprising amino acid sequences corresponding to a chemokine and a hapten that are useful as vaccines. The polypeptides of the present invention may include a hapten that is a Meningitis Related Homologous Antigenic 10 Sequences (MRHAS) from a bacterial or viral agent known to cause meningitis. These peptides induce protective immunity in a host susceptible to meningitis. The present invention also relates to materials useful in the diagnosis of diseases, including meningitis, by providing monoclonal antibodies, peptides, and mixtures and combinations thereof, that are useful in detection of disease-causing organisms.
2. Meningitis
The term “meningitis” is a general one, referring to the inflammatory response to infection of the meninges and the cerebrospinal fluid (CSF). See Roos, “Chapter 16”, in Scheld, et al., eds., 1991, Infections of the Central Nervous System: 335-403.
The fact that the inflammatory response occurs in the proximity of the brain and in the space limited by a rigid cranium, makes these infections serious and life threatening. Most patients exhibit nonspecific clinical signs and symptoms such as fever, irritability, altered mental status usually accompanied by vomiting and loss of appetite. In children one year of age and older, photophobia and headache are common complaints. Specific clinical signs indicative of meningitis are neck rigidity and pain on neck flexion. Brudzinski's sign (neck flexion producing knee and hip flexion) and Kernig's sign (difficulty and pain in raising extended leg) are other Useful clinical signs.
In infants less than 6 months old, early diagnosis of meningitis is difficult because signs of meningitis are not prominent and neck rigidity is often absent. Such patients commonly exhibit fever, respiratory distress, other signs of sepsis, and convulsions. Bulging anterior fontanelle due to increased intracranial pressure may be the only specific sign.
Petechiae (or rash) is, most commonly present in meningococcal infections. In severe meningococcal infections, bacteremia, petechiae and shock may develop with alarming rapidity. Convulsions at some point in the illness occur in about 30% of the cases. This number is 20 often higher in neonates and infants under one year of age. Other acute complications include septic shock, disseminated intravascular coagulation, syndrome of inappropriate antidiuretic hormone, increased intracranial pressure, and diabetes ins ipidus. 25 Convulsions and coma appearing with 24 hours accompanied by high fever indicates serious infection. Stutman & Marks, 1987, Clin. Ped., 26:432-438.
A diverse array of both bacteria and viruses cause meningitis, the infectivity of which is dependent on a complex array of factors, including virulence of the organisms, the carrier state, and the host's humoral immune response.
Viral Causes of Meningitis
Viruses generally cause milder forms of meningitis 35 (e.g. meningomyelitis and aseptic meningitis) with a short clinical course and reduced mortality. Agents most commonly associated are coxsackievirus A (types 2,4,7,9,10), B (types 1-6), polio virus, echoviruses (types 1-34, except, 12, 24, 26, 29, 32-34), enteroviruses (types 70, 71), human immunodeficiency virus-1 (HIV-1), and rubella virus (RV). See Melnick, “Chapter 33” and Cooper, “Chapter 42” in Fields, et al., eds., 1985 5 Virology: 739-794 and 1005-1032, respectively; and Rotbart, “Chapter 3”, in Scheld et al., 1991, infra: 19-33.
Rubella is possibly the most common cause of viral meningitis. Rubella is a highly contagious disease, usually associated with childhood, and is characterized by a general rash and a mild fever. Sub-clinical infections are also common. Its clinical aspects have been confused with measles, which it closely resembles. The infection of a pregnant woman poses the greatest risk when infection of the fetus can lead to spontaneous abortion or an array of abnormalities called the Congenital Rubella Syndrome in the newborn. Damage most frequently involves cardiac abnormalities, deafness, cataracts, blindness and Central Nervous System (CNS) 20 disorders including microencephaly.
The rubella virion is a spherical, enveloped virus, approximately 60 rim in diameter, and is a member of the Togaviridae. The RV genome is a 10 Kb plus single-stranded RNA. The outer envelope is comprised of 25 lipoproteins derived from the infected host cell, and it appears to have two viral encoded glycoproteins, E1 (58 Kd) and E2 (42-47 Kd), responsible for the hemagglutination activity of the virus. Its core protein is a non-glycosylated nucleocapsid protein with an 30 approximate weight of 33 Kd. It appears that the core; E1, and E2 are all derived from the same parent protein or structural polyprotein. See Clark et al., 1987, Nucl. Acids Res., 15:3041-3057; Dominguez, et al., 1990, Virology, 177:225-238. Three strains of wild type RV 35 (M33, Therien, Judith) and a vaccine strain (TPV77) of RV have been identified and sequenced (Zheng et al., 1988, Arch, Viral., 98:189-197). Between these different wild type strains, there exists minor variations in the amino acid sequence of the structural polyprotein.
The detection of RV in diagnosis has in the past proven difficult, largely because the virus grows to low titers in the tissue cultures and is highly labile, making it technically difficult to isolate and purify (Ho-Terry et al., 1986, Arch. ViroI., 87:219-228).
The detection of RV in the CNS presents additional technical problems. It has been known since 1941 that the RV can infect cells of the CNS (Gregg, 1941, Trans. Ophthalmol. Soc. Aust., 3:3546). However, it has proven difficult to reliably demonstrate the presence of the RV in infected brain tissue. Persistent infection of the CNS has been well documented in the congenital rubella syndrome (Desmond et al., 1967, J. Pediat., 7:311-331), and in the neuropathology of progressive rubella panencephalitis of late onset occurs where the virus has been isolated from brain biopsy material (Townsend et al., 1975, N. Engl. J. Med., 292:990-993; Cremer et al., 1979, J. Gen. Virol., 29:143-153). Less commonly documented are the wide range of neuropathies known to follow exposure to V. These include encephalitis, meningomyelitis, and bilateral optic neuritis (Connolly et al., 1975, Brain, 98:583-594). Moreover, the report of a diffuse myelitis following RV in cells of the nervous system requires further investigation (Holt et al. 1975, Brit. Med. J., 7:1037-1038).
RV-directed polypeptide synthesis in normal rat glial cells in continuous tissue culture has been studied •(Singh & Van Alstyne, 1978, Brain Res., 155:418-421). Unlike a productive rubella virus infection in permissive murine L (muscle) cells, infection of normal glial cells resulted in no detectable progeny virions in tissue culture supernatants and no detectable rubella 33 Kd core protein in infected cell lysates (Pope and Van Alstyne, 1981, Virology, 124:173-180). Furthermore, exposure of infected glial cells to dibutyryl cyclic adenine monophosphate reversed the restriction, resulting in the appearance of the 33 Kd rubella nucleocapsid protein in infected cell lysates and the appearance of mature progeny virions in tissue culture supernatants (Van Alstyne and Paty, 1983, Virology, 124:173-180).
Early diagnostic tests were based on the hemagglutinating properties of its external glycoproteins. Commonly, the hemagglutination inhibition assays relied on the presence of antibodies to the RV hemagglutinin (HA) in the serum samples to inhibit the viral-mediated hemagglutination of chick red blood cells (Herrmann, “Rubella Virus”, 1979, in Diagnostic Procedures For Viral, Rickettsial And Chlamylial Infections, 725-766). The presence of high inhibition, indicated the indirect measurement of antibodies to the HA protein, and thereby, a recent rubella infection.
More recent tests employ enzyme-labelled antibodies in the enzyme-linked-immunosorbent assays (ELISA) (Voller & Biowell, 1975, Br. J. Exp. Pathol., 56:338-339), These assays are also indirect tests to measure the amount of circulating antibody to RV as an indication of infection. Indirect ELISA tests for RV employ bound viral antigens on a plastic microwells and the presence of bound antibodies linked to enzymes such as horseradish peroxidase.
There are several problems with the use of the indirect RV ELISA kits. These relate to low antibody titers observed with RV infection, the need for elaborate “cut-off” value calculations to eliminate background binding, the limited use of the test in the detection of low levels of specific viral antigens present in chronic CNS infection, and the tedious and time consuming nature of the test performance.
Furthermore, a live, attenuated rubella vaccine has been developed (Parkman et al., 1966, An. Engl. J. Med., 275:569-574). This vaccine is immunogenic in at least 95% of the recipients, and does confer protection against reinfection, in spite of the fact that it induces antibody levels which are significantly lower than those generated by wild type virus infection. However, a serious drawback associated with the administration of the attenuated vaccine is the significant proportion of adult females that go on to develop rubella-associated arthritis. Furthermore, recently immunized individuals still harbour infectious virus and are therefore infectious, proving dangerous to pregnant women with whom they may be in contact.
Another virus responsible for meningitis is the Human Immunodeficiency Virus-1 (HIV-1). HIV-1 is a human retrovirus which has been identified as the etiological agent of AIDS, an infectious and fatal disease transmitted through intimate sexual contact and exposure to contaminated blood or blood products. HIV-1 is related to the lentiviruses on the basis of its biological and in vitro characteristics, morphology and nucleotide sequences. It is also referred to as Human T cell Lymphotrophic Virus, type III, Lymphadenopathy Associated Virus, and AIDS Associated Retrovirus (Gallo, et al., 1984, Science, 224:500-503; Sarngadharan, et al., 1984, Science, 224:506-508; Barre-Sinoussi, et al., 1983, Science, 220:868-871; Levy, 1984, Science, 225:840-842; Gonda et al., 1985, Science, 227:177-179; Stephan, et al., 1986, Science, 231:589-594). Much interest has been focused on the effect of the long term, persistent infection of the immune system, by HIV-1. Recent information indicates that the virus moves from blood to the lymph nodes and thymus where it remains active, culminating in viremia, a precipitous drop in the CD4+ T-cell count, and one or more of the several symptoms known as AIDS.
However, primary HIV-1 infection itself results in an immediate set of defined clinical features. Commonly, an acute febrile illness resembling influenza or mononucleosis is noted. In addition, lymphocytic meningitis may accompany the febrile illness and the patient may then be presented with headache, stiff neck and photophobia, as well as rigors, arthralgias and myalgias, truncal maculopapular rash, urticaria, abdominal cramps and diarrhea (Ho, 1985, Ann. Internal Medicine, 103:880-883).
While some patients remain asymptomatic for up to 3 months preceding their seroconversion, indicating that HIV-1 infection may be subclinical, primary infection should be included in the differential diagnosis of prolonged febrile illnesses in persons at risk for AIDS. The presence of a maculopapular or urticarial rash, or lymphocytic meningitis is compatible with this diagnosis. Hence, early recognition of the varied syndromes associated with this virus might permit effective treatment before immunologic abnormalities become established.
Currently, one of the most commonly used direct tests for HIV-1 infection employs the following approaches: (i) direct culturing of virus from infected blood or blood cells and subsequent in vitro, propagation of the virus in lymphocyte cultures; (ii) measuring reverse transcriptase levels; (iii) immunocytochemical staining of viral proteins; (iv) electron microscopy; (v) hybridization of nucleic acid probes; and measuring HIV-1 antigens with enzyme immunoassays (Goudsmit et al., 1986, Brit. Med. 2993:1459-1462; Caruso et al., 1987, J. Virol. Methods, 17:199-210).
HIV-1 appears to have at least three core proteins (p17, p24, and p15) that are derived from a core polyprotein called gag polyprotein. See Muesing, et al., 1985, Nature, 313:450-458. The gag polyprotein in the LV isolate of HIV-1 is 478 amino acids long and the three mature core proteins appear to be derived as p17 from amino acid sequence numbers 1-132, p24 from amino acid 30 sequence numbers 133-391, and p15 from amino acid sequence numbers 392-478 (Muesing, infra). Moreover, it appears that the HIV-1 (LAV-1a isolate) also has at least one capsid transmembrane glycoprotein derived from a 861-amino acid long Envelope Polyprotein (Wain-Hobson, et al., 1985, Cell, 40:9-17).
Enzyme immunoassays have clearly shown the diagnostic importance of the presence of the p24 core protein. A correlation has been established between viremia, the decline of antibodies to p24, and the progression of symptoms from the asymptomatic seropositivity to fully expressed AIDS (Lange et al., 1986, Brit. Med. J., 293:1459-1462; Paul et al., 1987, J. Med. Virol., 22:357-363; Forster et al., 1987 AIDS, 1:235-240). A, decline in the p24 level has also been observed to occur inpatients treated with AZT (Chaisson et al., 1986, New Eng. J. Med., 315:1610-1611).
Assays for the direct detection of p24 are currently on the market (Allain, infra; Forster, infra). These assays use the same sandwich format in which serum samples are incubated with bound and enzyme-labelled anti-p24-antibodies to form an antibody/p24-antigen-antibody sandwich. Antigen levels of approximately 50 picograms/ml can be detected, when the antigen concentration is read from a, standard curve constructed with a set of p24 standards of known concentrations. The tests are tedious and time consuming to perform, require dilutions of patients' sera, and do not provide information regarding the comparisons of rising antigen and concomitant declining antibody levels necessary to evaluate laboratory findings.
There are significant difficulties inherent in designing a vaccine which will confer protection against HIV-1. The vaccine must differentiate between HIV-1 and closely-related virus, HIV-2. The rapid rate of HIV-1 mutation requires that the antigen(s) be highly conserved. Moreover, the HIV-1 infection of a small subset of T cells requires the killing of an integral part of the immune cell network, with unknown consequences, to completely eradicate the virus. In addition, vaccinated antigens could enter lymph nodes and stimulate B cells to produce cytokines that in turn stimulate HIV-1 infection of T cells, and thereby having a reverse effect, causing a more rapid onset of AIDS.
Peptides from gp120, gp160, gp41, gp120 +gp41, p17 and p14 are currently being employed for vaccine production by several companies and universities (Spalding, 1992, Biotech., 10:24-29.) However, these peptides are being tested for their ability to solely induce B cells to produce neutralizing antibody.